The present invention relates to fluorescent labels that can be attached to nucleotide derivatives for use in nucleic acid sequencing. The nucleotide derivatives are nucleic acid chain terminating substrates that when attached to a fluorescent label afford fluorescently labeled chain terminators. These fluorescently labeled chain terminators can be incorporated into nucleic acid fragments, preferably the 3′ terminal end, to identify the sequence of a nucleic acid. To be useful as a nucleic acid chain terminator substrate in fluorescence based nucleic acid sequencing, the chain terminator substrate must contain a fluorescent reporter and a nucleotide derivative that is capable of being added to a nucleic acid sequence, but is not capable of being used by a replication enzyme to attach a subsequent nucleotide or nucleotide derivative to the nucleic acid sequence.
Cyanine dyes used to detect biomolecules and in particular as a fluorescent reporter for labeling nucleic acid chain terminator substrates are known. However, these compounds can interfere with the binding or interaction of the nucleotide derivative with the replication enzyme, are unstable, difficult to synthetically manufacture, or have a fluorescent detection wavelength that is problematic for automated systems. Further information on fluorescent probes used to detect biomolecules can be found in Flanagan, J. H., et al., Bioconjugate Chem. 8:751-756 (1997); Mujumdar, R. B., et al., Bioconjugate Chem., 4:2 105-111 (1993); Mujumdar, R. B., et al., Cytometry, 10:11-19 (1989); Mujumdar, S. R., et al., Bioconjugate Chem., 7:356-362 (1996); Ozmen, B., et al., Tetrahedron Letters, 41:9185-9188 (2000); Shealy, D. B., et al., Anal. Chem. 67:247-251 (1995); Southwick, P. L., et al., Cytometry, 11418-430 (1990); Strekowski, L., et al., J. Org. Chem., 57:4578-4580 (1992); and Williams, R. J., et al., Anal. Chem., 65:601-605 (1993); and U.S. Pat. Nos. 5,453,505; 5,571,388; and 6,002,003.
Therefore, there is a need for a fluorescently labeled nucleic acid chain terminator that does not interfere with nucleotide replication, that is stable, and has a fluorescent detection wavelength that is amenable to automated systems.